An Engineered Glycerol Dehydratase With Improved Activity for the Conversion of meso‐2, 3‐butanediol to Butanone. Issue 12 (25th September 2017)

• Record Type: Journal Article
• Authors: Maddock, Danielle J.; Gerth, Monica L.; Patrick, Wayne M.
• Is Part Of: Biotechnology journal. Volume 12:Issue 12(2017)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2017-09-25
• Subjects: butanone -- combinatorial active site saturation test -- ConSurf -- fusion protein -- Klebsiella pneumoniae glycerol dehydratase; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.201700480
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract : There is substantial interest in engineering microorganisms to produce industrial chemicals that are currently derived from petroleum. One of these petrochemicals is butanone, which could be produced from microbially synthesized 2, 3‐butanediol through the action of a suitable dehydratase enzyme. Unfortunately, however, there are no known enzymes that natively catalyze this reaction. In this work, the authors set out to engineer the B12 ‐dependent glycerol dehydratase from Klebsiella pneumoniae ( Kp GDHt), in order to increase its activity for the conversion of meso ‐2, 3‐butanediol into butanone. The authors began by fusing the α and β subunits of the enzyme, to simplify downstream high‐throughput screening protocols. Serendipitously, the fusion protein showed a 20°C increase in its temperature optimum. Using this stabilized scaffold as a starting point, the authors employed the combinatorial active site saturation test and consensus‐guided mutagenesis to randomize 28 residues within 12 Å of the Kp GDHt active site. By screening over 5500 variants, the authors discovered a single point mutation (T200S) that increased the catalytic efficiency of meso ‐2, 3‐butanediol dehydration by four‐fold, to a value of k cat / K M = 5.1 × 10 3 M −1 s −1 . Thus the authors report what is, to date, the most comprehensive mutagenesis and the largest engineered increase in catalytic efficiency on the B12 ‐dependent glycerol dehydratase scaffold. Abstract : There is substantial interest in engineering microorganisms to produce the industrial chemicals that are currently derived from petroleum. One such chemical is butanone, which is a component of paints, coatings, adhesives, inks, and pharmaceuticals. In this study, the authors engineered a bacterial enzyme to be four‐fold better at producing butanone from a biologically‐derived building block.